Gene alterations in the nuclear transport receptor superfamily: A study of head and neck cancer.

In cancer cells, the nuclear transport system is often disrupted, leading to abnormal localization of nuclear proteins and altered gene expression. This disruption can arise from various mechanisms such as mutations in genes that regulate nuclear transport, altered expression of transport proteins, and changes in nuclear envelope structure. Oncogenic protein build-up in the nucleus due to the disturbance in nuclear transport can also boost tumor growth and cell proliferation. In this study, we performed bioinformatic analyses of 23 key nuclear transport receptors using genomic and transcriptomic data from pancancer and head and neck squamous cell carcinoma (HNSCC) datasets from The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia and found that the total alteration frequency of 23 nuclear transport receptors in 2691 samples of the PCAWG Consortium was 42.1% and a high levels of genetic alterations was significantly associated with poor overall survival. Amplification was the most common type of genetic alterations, and results in the overexpression of nuclear transport receptors in HNSCC compared to normal tissues. Furthermore, our study revealed that seven out of eight cell cycle genes (CDK1, CDK2, CDK4, CDK6, CCNA1, CCNB1, and CCNE2) were significantly and positively correlated with nuclear transport receptor genes in TCGA pancancer and CCLE datasets. Additionally, functional enrichment analysis showed that nuclear transport receptor genes were mainly enriched in the adhesion junction, cell cycle, ERBB, MAPK, MTOR and WNT signaling pathways.

Reliability of online dental final exams in the pre and post COVID-19 era: A comparative study.

Amidst the fourth COVID-19 wave in Viet Nam, national lockdowns necessitated the closure of numerous dental schools. To assess DDS (Doctor of Dental Surgery) graduation exams, this study analyzed their 2021 implementation in comparison to onsite exams conducted in 2020 and 2022 at the Faculty of Odonto-Stomatology, University of Medicine and Pharmacy at Ho Chi Minh City, Viet Nam (FOS-UMPH). The final online examination comprises two main sessions: a synchronous online examination using FOS-UMPH e-Learning for theories (consisting of 200 MCQs and 3 written tests with 3 clinical situations needed be solved) and a synchronous online examination using Microsoft Teams for practicum (comprising of 12 online OSCE stations). The final grades were evaluated using the same metrics in face-to-face final examinations in 2022 and 2020. A total of 114, 112 and 95 students were recruited for the first-time exams in 2020, 2021 and 2022, respectively. In order to analyze the reliability, histogram and k-mean clustering were employed. The histograms from 2020, 2021 and 2022 showed a striking similarity. However, fewer students failed in 2021 and 2022 (13% and 12.6%, respectively) compared to 2020 (28%), with clinical problem-solving part grades (belonging to theory session) being notably higher in 2021 and 2022. Intriguingly, the MCQ Score results showed the identical patterns. The courses of orthodontics, dental public health, and pediatrics subjects (in the group of prevention and development dentistry) stood out for their exceptional accuracy across both sessions. After examining data gathered over three years, we identified three distinct clusters: the first comprised of scattered average and low scores, the second characterized by high scores but unstable and scattered and the third cluster boasting consistently high and centered scores. According to our study, online and onsite traditional graduation exam results are relatively equivalent, but additional measures are necessary to standardize the final examination and adapt to the new normal trend in dental education.

Periodontitis promotes the expression of gingival transmembrane serine protease 2 (TMPRSS2), a priming protease for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

OBJECTIVES: The oral cavity is one of the main entry sites for SARS-CoV-2. Gingival keratinocytes express transmembrane serine protease 2 (TMPRSS2), responsible for priming the SARS-CoV-2 spike protein. We investigated whether periodontitis increased the expression of TMPRSS2. METHODS: To investigate gene expression in periodontitis, we analyzed the expression of specific genes from (1) the Gene Expression Omnibus (GEO) dataset of 247 human gingival tissues and (2) an experimentally-induced periodontitis mouse model. Human gingival tissues with or without periodontitis were immunohistochemically stained using an anti-TMPRSS2 antibody. Analysis of the TMPRSS2 promoter was performed using a ChIP-Atlas dataset. TMPRSS2 expression was detected in cultured human keratinocytes using quantitative reverse transcription (qRT)-PCR and Western blot analysis. RESULTS: GEO dataset analysis and an experimentally-induced periodontitis model revealed increased expression of TMPRSS2 in periodontitis gingiva. The keratinocyte cell membrane in periodontitis gingiva was strongly immunohistochemically stained for TMPRSS2. Using ChIP-Atlas and GEO datasets, we screened for transcription factors that bind to the TMPRSS2 promoter region. We found one candidate, estrogen receptor 1 (ESR1), highly expressed in periodontitis gingiva. Analysis of the GEO dataset revealed a correlation between ESR1 and TMPRSS2 expression in gingival tissues. An ESR1 ligand induced TMPRSS2 expression in cultured keratinocytes. CONCLUSIONS: Periodontitis increases TMPRSS2 expression in the cell membrane of gingival keratinocytes.

Searching for New Molecular Targets for Oral Squamous Cell Carcinoma with a View to Clinical Implementation of Precision Medicine.

Head and neck cancer, including oral squamous cell carcinoma (OSCC), is the eighth most common malignancy globally and is characterized by local invasiveness and high nodal metastatic potential. The OSCC incidence is also increasing, and the number of deaths is also rising steadily in Japan. The development of molecular markers to eradicate OSCC is an urgent issue for humankind. The increase in OSCC despite the declining smoking rate may be due to several viral infections through various sexual activities and the involvement of previously unfocused carcinogens, and genetic alterations in individual patients are considered to be more complicated. Given this situation, it is difficult to combat OSCC with conventional radiotherapy and chemotherapy using cell-killing anticancer drugs alone, and the development of precision medicine, which aims to provide tailor-made medicine based on the genetic background of each patient, is gaining attention. In this review article, the current status of the comprehensive search for driver genes and biomarkers in OSCC will be briefly described, and some of the candidates for novel markers of OSCC that were found will be outlined.

IFN-γ‒Induced APOBEC3B Contributes to Merkel Cell Polyomavirus Genome Mutagenesis in Merkel Cell Carcinoma.

Merkel cell polyomavirus (MCPyV) is the causative agent of an aggressive skin tumor, Merkel cell carcinoma. The viral genome is integrated into the tumor genome and harbors nonsense mutations in the helicase domain of large T antigen. However, the molecular mechanisms by which the viral genome gains the tumor-specific mutations remain to be elucidated. Focusing on host cytosine deaminases APOBEC3s, we find that A3A, A3B, or A3G introduces A3-specific mutations into episomal MCPyV genomes in MCPyV-replicating 293-derivative cells. Sequence analysis of MCPyV genomes retrieved from the NCBI database revealed a decrease of TpC dinucleotide, a preferred target for A3A and A3B, in the 3'-region of the large T antigen‒coding sequence. The viral DNA isolated from tumors contained mutated cytosines, with a remarkable bias toward TpC dinucleotide. Analysis of publicly available microarray data showed that expression of IFN-γ and cytotoxic T lymphocyte markers was positively correlated with the A3A, A3B, and A3G levels in MCPyV-positive but not in MCPyV-negative tumors. Finally, IFN-γ treatment induced A3B and A3G expression in the MCPyV-positive Merkel cell carcinoma cell line MS-1. These results suggest that the IFN-γ-A3B axis plays pivotal roles in evolutionally shaping MCPyV genomic sequences and in generating tumor-specific large T antigen mutations during development of Merkel cell carcinoma.

Atypical immunohistochemical patterns can complement the histopathological diagnosis of oral premalignant lesions.

BACKGROUND: The histopathological diagnosis of oral premalignant lesions (OPLs) such as oral epithelial dysplasia (ED) and carcinoma in situ (CIS), as well as epithelial hyperplasia (EHP), is important for the early detection and precise treatment of oral squamous cell carcinoma. However, the standardization of detection and treatment by histological criteria alone remains challenging owing to the complicated and varied histology. We evaluated practically useful immunohistochemical (IHC) markers that might complement the histopathological diagnosis of OPLs. METHODS: We re-evaluated the histopathological diagnoses and IHC patterns of Ki-67, TP53, CK13, and CK17 in 200 cases of OPLs and performed multiple logistic regression analyses for their predictive accuracy. RESULTS: We identified and compared atypical IHC patterns in OPLs and in the normal epithelium. Ki-67 expression showed specific patterns in categorized OPLs as EHP, low-grade dysplasia (LED), high-grade dysplasia (HED), and CIS. Multiple logistic regression analyses in the quadrant categories revealed that EHP and CIS had high predictive accuracies of 90.1% and 96.2%, respectively, and in binary categories, combined EHP and LED versus combined HED and CIS showed predictive accuracies of 92.1% and 89.9%, respectively. Binominal logistic regression analysis between each quadrant category revealed satisfactory predictive accuracy of EHP vs. LED, LED vs. HED, and HED vs. CIS (75.2%, 78.9%, and 87.9%, respectively), and Ki-67 showed the highest adjusted odds ratio, followed by TP53. CONCLUSION: The proposed atypical IHC patterns might serve as useful markers to supplement the morphological diagnosis of OPLs, and established IHC methods for Ki-67 and TP53 might provide stable results.

CXCL13 is a differentiation- and hypoxia-induced adipocytokine that exacerbates the inflammatory phenotype of adipocytes through PHLPP1 induction.

Hypoxia in adipose tissue is regarded as a trigger that induces dysregulation of the secretory profile in adipocytes. Similarly, local dysregulation of adipocytokine secretion is an initial event in the deleterious effects of obesity on metabolism. We previously reported that CXCL13 is highly produced during adipogenesis, however little is known about the roles of CXCL13 in adipocytes. Here, we found that hypoxia, as modeled by 1% O2 or exposure to the hypoxia-mimetic reagent desferrioxamine (DFO) has strong inductive effects on the expression of CXCL13 and CXCR5, a CXCL13 receptor, in both undifferentiated and differentiated adipocytes and in organ-cultured white adipose tissue (WAT). CXCL13 was also highly expressed in WAT from high fat diet-fed mice. Hypoxic profile, typified by increased expression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) and decreased expression of adiponectin, was significantly induced by CXCL13 treatment during adipogenic differentiation. Conversely, the treatment of adipocytes with a neutralizing-antibody against CXCL13 as well as CXCR5 knockdown by specific siRNA effectively inhibited DFO-induced inflammation. The phosphorylation of Akt2, a protective factor of adipose inflammation, was significantly inhibited by CXCL13 treatment during adipogenic differentiation. Mechanistically, CXCL13 induces the expression of PHLPP1, an Akt2 phosphatase, through focal adhesion kinase (FAK) signaling; and correspondingly we show that CXCL13 and DFO-induced IL-6 and PAI-1 expression was blocked by Phlpp1 knockdown. Furthermore, we revealed the functional binding sites of PPARγ2 and HIF1-α within the Cxcl13 promoter. Taken together, these results indicate that CXCL13 is an adipocytokine that facilitates hypoxia-induced inflammation in adipocytes through FAK-mediated induction of PHLPP1 in autocrine and/or paracrine manner.

BMP9 prevents induction of osteopontin in JNK-inactivated osteoblasts via Hey1-Id4 interaction.

Osteopontin (OPN) is an osteoblast-derived secretory protein that plays a role in bone remodeling, osteoblast responsiveness, and inflammation. We recently found that osteoblast differentiation is type-specific, with conditions of JNK inactivation inducing osteoblasts that preferentially express OPN (OPN-type). Since OPN-type osteoblasts highly express osteogenesis-inhibiting proteins and Rankl, an important inducer of osteoclastogenesis, an increased appearance of OPN-type osteoblasts may be associated with inefficient and poor-quality bone regeneration. However, whether specific osteogenic inducers can modulate OPN-type osteoblast differentiation is completely unknown. Here, we demonstrate that bone morphogenic protein 9 (BMP9) prevents induction of OPN-type osteoblast differentiation under conditions of JNK inhibition. Although JNK inactivation suppressed both BMP2- and BMP9-induced matrix mineralization and osteocalcin expression, the expression of Rankl and specific cytokines such as Gpha2, Esm1, and Sfrp1 under similar conditions was increased in all cells except those treated with BMP9. Increased expression of Id4, a critical transcriptional regulator of OPN-type osteoblast differentiation, was similarly prevented only in BMP9-treated cells. We also found that BMP9 specifically induces the expression of Hey1, a bHLH transcriptional repressor, and that Id4 inhibits the suppressive effects of Hey1 on Opn promoter activity by forming Id4-Hey1 complexes in osteoblasts. Using site-direct mutagenesis, ChIP, and immunoprecipitation, we elucidated that BMP9-induced overexpression of Hey1 can overcome the effects of Id4 and suppress OPN expression. We further found that p38 activation and JNK inactivation are involved in BMP9-induced Hey1 expression. Collectively, these data suggest that BMP9 is a unique osteogenic inducer that regulates OPN-type osteoblast differentiation.

Low intensity pulsed ultrasound (LIPUS) maintains osteogenic potency by the increased expression and stability of Nanog through spleen tyrosine kinase (Syk) activation.

Mesenchymal stem cells (MSCs) are a powerful tool for cell-based, clinical therapies like bone regeneration. Therapeutic use of cell transplantation requires many cells, however, the expansion process needed to produce large quantities of cells reduces the differentiation potential of MSCs. Here, we examined the protective effects of low intensity pulsed ultrasound (LIPUS) on the maintenance of osteogenic potency. Primary osteoblastic cells were serially passaged between 2 and 12 times with daily LIPUS treatment. We found that LIPUS stimulation maintains osteogenic differentiation capacity in serially passaged cells, as characterized by improved matrix mineralization and Osteocalcin mRNA expression. Decreased expression of Nanog, Sox2, and Msx2, and increased expression of Pparg2 from serial passaging was recovered in LIPUS-stimulated cells. We found that LIPUS stimulation not only increased but also sustained expression of Nanog in primary osteoblasts and ST2 cells, a mouse mesenchymal stromal cell line. Nanog overexpression in serially passaged cells mimicked the recuperative effects of LIPUS on osteogenic potency, highlighting the important role of Nanog in LIPUS stimulation. Additionally, we found that spleen tyrosine kinase (Syk) is an important signaling molecule to induce Nanog expression in LIPUS-stimulated cells. Syk activation was regulated by both Rho-associated kinase 1 (ROCK1) and extracellular ATP in a paracrine manner. Interestingly, the LIPUS-induced increase in Nanog mRNA expression was regulated by ATP-P2X4-Syk Y323 activation, while the improvement of Nanog protein stability was controlled by the ROCK1-Syk Y525/526 pathway. Taken together, these results indicate that LIPUS stimulation recovers and maintains the osteogenic potency of serially passaged cells through a Syk-Nanog axis.

Continuity of transcriptomes among colorectal cancer subtypes based on meta-analysis.

BACKGROUND: Previous approaches to defining subtypes of colorectal carcinoma (CRC) and other cancers based on transcriptomes have assumed the existence of discrete subtypes. We analyze gene expression patterns of colorectal tumors from a large number of patients to test this assumption and propose an approach to identify potentially a continuum of subtypes that are present across independent studies and cohorts. RESULTS: We examine the assumption of discrete CRC subtypes by integrating 18 published gene expression datasets and > 3700 patients, and contrary to previous reports, find no evidence to support the existence of discrete transcriptional subtypes. Using a meta-analysis approach to identify co-expression patterns present in multiple datasets, we identify and define robust, continuously varying subtype scores to represent CRC transcriptomes. The subtype scores are consistent with established subtypes (including microsatellite instability and previously proposed discrete transcriptome subtypes), but better represent overall transcriptional activity than do discrete subtypes. The scores are also better predictors of tumor location, stage, grade, and times of disease-free survival than discrete subtypes. Gene set enrichment analysis reveals that the subtype scores characterize T-cell function, inflammation response, and cyclin-dependent kinase regulation of DNA replication. CONCLUSIONS: We find no evidence to support discrete subtypes of the CRC transcriptome and instead propose two validated scores to better characterize a continuity of CRC transcriptomes.

SMO expression in colorectal cancer: associations with clinical, pathological, and molecular features.

BACKGROUND: Smoothened, frizzled family receptor (SMO) is an important component of the hedgehog signaling pathway, which has been implicated in various human carcinomas. However, clinical, molecular, and prognostic associations of SMO expression in colorectal cancer remain unclear. METHODS: Using a database of 735 colon and rectal cancers in the Nurse's Health Study and the Health Professionals Follow-up Study, we examined the relationship of tumor SMO expression (assessed by immunohistochemistry) to prognosis, and to clinical, pathological, and tumor molecular features, including mutations of KRAS, BRAF, and PIK3CA, microsatellite instability, CpG island methylator phenotype (CIMP), LINE-1 methylation, and expression of phosphorylated AKT and CTNNB1. RESULTS: SMO expression was detected in 370 tumors (50 %). In multivariate logistic regression analysis, SMO expression was independently inversely associated with phosphorylated AKT expression [odds ratio (OR) 0.48; 95 % confidence interval (CI) 0.34-0.67] and CTNNB1 nuclear localization (OR 0.48; 95 % CI 0.35-0.67). SMO expression was not significantly associated with colorectal cancer-specific or overall survival. However, in CIMP-high tumors, but not CIMP-low/0 tumors, SMO expression was significantly associated with better colorectal cancer-specific survival (log-rank P = 0.012; multivariate hazard ratio, 0.36; 95 % CI 0.13-0.95; P interaction = 0.035, for SMO and CIMP status). CONCLUSIONS: Our data reveal novel potential associations between the hedgehog, the WNT/CTNNB1, and the PI3K (phosphatidylinositol-4,5-bisphosphonate 3-kinase)/AKT pathways, supporting pivotal roles of SMO and hedgehog signaling in pathway networking. SMO expression in colorectal cancer may interact with tumor CIMP status to affect patient prognosis, although confirmation by future studies is needed.

A prospective study of duration of smoking cessation and colorectal cancer risk by epigenetics-related tumor classification.

The effect of duration of cigarette smoking cessation on colorectal cancer risk by molecular subtypes remains unclear. Using duplication-method Cox proportional-hazards regression analyses, we examined associations between duration of smoking cessation and colorectal cancer risk according to status of CpG island methylator phenotype (CIMP), microsatellite instability, v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation, or DNA methyltransferase-3B (DNMT3B) expression. Follow-up of 134,204 individuals in 2 US nationwide prospective cohorts (Nurses' Health Study (1980-2008) and Health Professionals Follow-up Study (1986-2008)) resulted in 1,260 incident rectal and colon cancers with available molecular data. Compared with current smoking, 10-19, 20-39, and ≥40 years of smoking cessation were associated with a lower risk of CIMP-high colorectal cancer, with multivariate hazard ratios (95% confidence intervals) of 0.53 (0.29, 0.95), 0.52 (0.32, 0.85), and 0.50 (0.27, 0.94), respectively (Ptrend = 0.001), but not with the risk of CIMP-low/CIMP-negative cancer (Ptrend = 0.25) (Pheterogeneity = 0.02, between CIMP-high and CIMP-low/CIMP-negative cancer risks). Differential associations between smoking cessation and cancer risks by microsatellite instability (Pheterogeneity = 0.02), DNMT3B expression (Pheterogeneity = 0.03), and BRAF (Pheterogeneity = 0.10) status appeared to be driven by the associations of CIMP-high cancer with microsatellite instability-high, DNMT3B-positive, and BRAF-mutated cancers. These molecular pathological epidemiology data suggest a protective effect of smoking cessation on a DNA methylation-related carcinogenesis pathway leading to CIMP-high colorectal cancer.

Physical activity, tumor PTGS2 expression, and survival in patients with colorectal cancer.

BACKGROUND: Higher levels of physical activity are associated with lower colorectal carcinoma incidence and mortality, perhaps through influencing energy balance, cellular prosta7 systemic inflammation. Although evidence suggests interactive effects of energetics, sedentary lifestyle, and tumor CTNNB1 (ß-catenin) or CDKN1B (p27) status on colon cancer prognosis, interactive effects of physical activity and tumor PTGS2 (the official symbol for COX-2) status on clinical outcome remain unknown. METHODS: Using molecular pathological epidemiology database of 605 stage I-III colon and rectal cancers in two prospective cohort studies (the Nurse's Health Study and the Health Professionals Follow-up Study), we examined patient survival according to postdiagnosis physical activity and tumor PTGS2 status (with 382 PTGS2-positive and 223 PTGS2-negative tumors by immunohistochemistry). Cox proportional hazards models were used to calculate colorectal cancer-specific mortality HR, adjusting for clinical and other tumor variables including microsatellite instability status. RESULTS: Among PTGS2-positive cases, compared with the least active first quartile, the multivariate HRs (95% confidence interval) were 0.30 (0.14-0.62) for the second, 0.38 (0.20-0.71) for the third, and 0.18 (0.08-0.41) for the fourth quartile of physical activity level (Ptrend = 0.0002). In contrast, among PTGS2-negative cases, physical activity level was not significantly associated with survival (Ptrend = 0.84; Pinteraction = 0.024, between physical activity and tumor PTGS2 status). CONCLUSIONS: Postdiagnosis physical activity is associated with better survival among patients with PTGS2-positive tumors but not among patients with PTGS2-negative tumors. IMPACT: Immunohistochemical PTGS2 expression in colorectal carcinoma may serve as a predictive biomarker in pathology practice, which may predict stronger benefit from exercise.

Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress.

Altered RNA processing is an underlying mechanism of amyotrophic lateral sclerosis (ALS). Missense mutations in a number of genes involved in RNA function and metabolisms are associated with ALS. Among these genes is angiogenin (ANG), the fifth member of the vertebrate-specific, secreted ribonuclease superfamily. ANG is an angiogenic ribonuclease, and both its angiogenic and ribonucleolytic activities are important for motor neuron health. Ribonuclease 4 (RNASE4), the fourth member of this superfamily, shares the same promoters with ANG and is co-expressed with ANG. However, the biological role of RNASE4 is unknown. To determine whether RNASE4 is involved in ALS pathogenesis, we sequenced the coding region of RNASE4 in ALS and control subjects and characterized the angiogenic, neurogenic, and neuroprotective activities of RNASE4 protein. We identified an allelic association of SNP rs3748338 with ALS and demonstrated that RNASE4 protein is able to induce angiogenesis in in vitro, ex vivo, and in vivo assays. RNASE4 also induces neural differentiation of P19 mouse embryonal carcinoma cells and mouse embryonic stem cells. Moreover, RNASE4 not only stimulates the formation of neurofilaments from mouse embryonic cortical neurons, but also protects hypothermia-induced degeneration. Importantly, systemic treatment with RNASE4 protein slowed weight loss and enhanced neuromuscular function of SOD1 (G93A) mice.

Expression profiling of archival tumors for long-term health studies.

PURPOSE: More than 20 million archival tissue samples are stored annually in the United States as formalin-fixed, paraffin-embedded (FFPE) blocks, but RNA degradation during fixation and storage has prevented their use for transcriptional profiling. New and highly sensitive assays for whole-transcriptome microarray analysis of FFPE tissues are now available, but resulting data include noise and variability for which previous expression array methods are inadequate. EXPERIMENTAL DESIGN: We present the two largest whole-genome expression studies from FFPE tissues to date, comprising 1,003 colorectal cancer (CRC) and 168 breast cancer samples, combined with a meta-analysis of 14 new and published FFPE microarray datasets. We develop and validate quality control (QC) methods through technical replication, independent samples, comparison to results from fresh-frozen tissue, and recovery of expected associations between gene expression and protein abundance. RESULTS: Archival tissues from large, multicenter studies showed a much wider range of transcriptional data quality relative to smaller or frozen tissue studies and required stringent QC for subsequent analysis. We developed novel methods for such QC of archival tissue expression profiles based on sample dynamic range and per-study median profile. This enabled validated identification of gene signatures of microsatellite instability and additional features of CRC, and improved recovery of associations between gene expression and protein abundance of MLH1, FASN, CDX2, MGMT, and SIRT1 in CRC tumors. CONCLUSIONS: These methods for large-scale QC of FFPE expression profiles enable study of the cancer transcriptome in relation to extensive clinicopathological information, tumor molecular biomarkers, and long-term lifestyle and outcome data.

Aspirin use, tumor PIK3CA mutation, and colorectal-cancer survival.

BACKGROUND: Regular use of aspirin after a diagnosis of colon cancer has been associated with a superior clinical outcome. Experimental evidence suggests that inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2) (also known as cyclooxygenase-2) by aspirin down-regulates phosphatidylinositol 3-kinase (PI3K) signaling activity. We hypothesized that the effect of aspirin on survival and prognosis in patients with cancers characterized by mutated PIK3CA (the phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene) might differ from the effect among those with wild-type PIK3CA cancers. METHODS: We obtained data on 964 patients with rectal or colon cancer from the Nurses' Health Study and the Health Professionals Follow-up Study, including data on aspirin use after diagnosis and the presence or absence of PIK3CA mutation. We used a Cox proportional-hazards model to compute the multivariate hazard ratio for death. We examined tumor markers, including PTGS2, phosphorylated AKT, KRAS, BRAF, microsatellite instability, CpG island methylator phenotype, and methylation of long interspersed nucleotide element 1. RESULTS: Among patients with mutated-PIK3CA colorectal cancers, regular use of aspirin after diagnosis was associated with superior colorectal cancer-specific survival (multivariate hazard ratio for cancer-related death, 0.18; 95% confidence interval [CI], 0.06 to 0.61; P<0.001 by the log-rank test) and overall survival (multivariate hazard ratio for death from any cause, 0.54; 95% CI, 0.31 to 0.94; P=0.01 by the log-rank test). In contrast, among patients with wild-type PIK3CA, regular use of aspirin after diagnosis was not associated with colorectal cancer-specific survival (multivariate hazard ratio, 0.96; 95% CI, 0.69 to 1.32; P=0.76 by the log-rank test; P=0.009 for interaction between aspirin and PIK3CA variables) or overall survival (multivariate hazard ratio, 0.94; 95% CI, 0.75 to 1.17; P=0.96 by the log-rank test; P=0.07 for interaction). CONCLUSIONS: Regular use of aspirin after diagnosis was associated with longer survival among patients with mutated-PIK3CA colorectal cancer, but not among patients with wild-type PIK3CA cancer. The findings from this molecular pathological epidemiology study suggest that the PIK3CA mutation in colorectal cancer may serve as a predictive molecular biomarker for adjuvant aspirin therapy. (Funded by The National Institutes of Health and others.).

No evidence for interference of h&e staining in DNA testing: usefulness of DNA extraction from H&E-stained archival tissue sections.

Although histochemical staining has been believed to inhibit the DNA amplification reaction, no previous study has systematically evaluated the influence of histochemical staining on downstream molecular assays. To evaluate an influence of H&E staining on DNA testing, we isolated DNA from 10 unstained, 10 hematoxylin-stained, 10 eosin-stained, and 10 H&E-stained tissue sections (ie, 4 groups), from each of 5 colon cancers. Among the 4 groups, we did not observe any significant or appreciable difference in DNA fragmentation by agarose gel electrophoresis, in DNA amplification by real-time polymerase chain reaction (PCR), in microsatellite PCR fragment analyses, or in a PCR-pyrosequencing assay. As a proof-of-principle study, we successfully performed microsatellite instability analysis and sequencing of KRAS and BRAF on more than 1,300 colorectal cancers using DNA extracted from H&E-stained tissue sections. Our data provide no evidence for an interfering effect of H&E staining on DNA testing, suggesting that DNA from H&E-stained sections can be effectively used for routine DNA testing.

Postmenopausal hormone therapy is associated with a reduced risk of colorectal cancer lacking CDKN1A expression.

Experimental studies have shown that estrogen- or progesterone-activated signaling leads to growth inhibition effects on colon cancer cells through the upregulation of several cell-cycle regulators. However, epidemiologic studies evaluating hormone therapy use and colorectal cancer risk by the status of cell-cycle regulators are lacking. In this study, we used data from the prospective Nurses' Health Study to evaluate whether the association between hormone therapy use and colorectal cancer risk differs by the molecular pathologic status of microsatellite instability (MSI) and expression of cell-cycle-related tumor biomarkers, including CDKN1A (p21, CIP1), CDKN1B (p27, KIP1), and TP53 (p53) by immunohistochemistry. Duplication Cox regression analysis was used to determine an association between hormone therapy use, cancer risk, and specific tumor biomarkers in 581 incident colon and rectal cancer cases that occurred during 26 years of follow-up among 105,520 postmenopausal women. We found a difference between hormone therapy use and colorectal cancer risk according to CDKN1A expression (P(heterogeneity) = 0.01). Current hormone therapy use was associated with a reduced risk for CDKN1A-nonexpressed [multivariate relative risk (RR), 0.61; 95% confidence interval (CI), 0.46-0.82] but not for CDKN1A-expressed (RR, 1.32; 95% CI, 0.76-2.31) tumors. The lower risk for CDKN1A-nonexpressed but not for CDKN1A-expressed cancers was also present among current users of estrogen-alone therapy. We found no significant difference in the relations between hormone therapy use and cancer risk according to MSI, CDKN1B, or TP53 status. Together, our molecular pathological epidemiology findings suggest a preventive effect of hormone therapy against colorectal carcinogenesis that depends, in part, on loss of cyclin-dependent kinase inhibitor CDKN1A.